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Abstrak :
Transfeksi merupakan proses memasukkan materi genetik ke dalam sel mamalia, yang umumnya digunakan untuk keperluan terapi gen dan produksi obat-obatan berbahan biologis (biologics) dari kultur sel mamalia. Pengantaran materi genetik melalui senyawa lipid (lipofeksi) merupakan metode transfeksi paling diminati karena sifatnya yang tidak toksik, mudah digunakan, terjangkau, dan memiliki nilai efisiensi transfeksi yang baik. Lipopeptida Pal-CK2H2-Tat(NLS)-TL(NLS) merupakan agen lipofeksi hasil pengembangan Badan Pengkajian dan Penerapan Teknologi (BPPT) yang diharapkan mampu menjadi kompetitor agen lipofeksi komersial yang sudah ada di pasar, akan tetapi kemampuannya dalam menginduksi produk biologis pada galur sel mamalia Chinese Hamster Ovary K1 (CHO-K1) belum terkarakterisasi dengan baik. Berkenaan dengan itu, proinsulin merupakan produk biologis bernilai tinggi yang pengembangannya juga sedang dilakukan oleh BPPT. Tujuan penelitian ini adalah mendapatkan klon plasmid proinsulin rekombinan serta mendeteksi produksi proinsulin hasil transfeksi plasmid yang menggunakan lipopeptida Pal-CK2H2-Tat(NLS)-TL(NLS) pada galur sel mamalia CHO-K1. Deteksi dilakukan melalui metode immunofluorescence assay (IFA) dan western blotting (WB). Hasil menunjukkan bahwa telah didapat 6 klon plasmid proinsulin rekombinan, dengan 1 klon terbukti fungsional karena mampu menginduksi ekspresi proinsulin dengan ukuran protein yang benar di dalam sel CHO-K1. Uji IFA dari hasil transfeksi yang menggunakan lipopeptida Pal-CK2H2-Tat(NLS)-TL(NLS) menunjukkan adanya perbedaan signifikan antara nilai Corrected Cell Total Fluorescence (CTCF) sel pasca transfeksi proinsulin rekombinan dan sel pasca transfeksi plasmid non-rekombinan pEGFP-N1. Uji pasca transfeksi yang menggunakan lipopeptida Pal-CK2H2-Tat(NLS)-TL(NLS) konsisten dengan pengujian IFA dan WB pasca transfeksi yang menggunakan agen lipofeksi komersial TurboFect™. Dapat disimpulkan bahwa protein proinsulin berhasil diekspresikan pasca transfeksi menggunakan lipopeptida Pal-CK2H2-Tat(NLS)-TL(NLS) pada galur sel mamalia CHO-K1. ......The process of introducing genetic materials into mammalian cells is known by transfection, which holds value in gene therapy and the production of biologics in mammalian cell culture. Among methods of transfection, deliverance through lipid particles or lipofection is the most favorable one because of several advantages. Pal-CK2H2-Tat(NLS)-TL(NLS) is a novel lipofection agent developed by the Agency for the Assessment and Application of Technology (BPPT) to compete with its successor in the field of lipofection, but its capability of inducing the production of biologics in CHO-K1 mammalian cell line was not characterized well. In the meantime, the same agency has been working on recombinant proinsulin, a highly valued biological product. The aims of this research are first to obtain clones of recombinant proinsulin and second to detect the presence of proinsulin in CHO-K1 cells post-transfected with recombinant proinsulin using Pal-CK2H2-Tat(NLS)-TL(NLS) lipopeptide. Protein detection is done through immunofluorescence assay (IFA) and western blotting (WB). Six plasmid clones of recombinant proinsulin have been obtained, with one clone proved to be functional in expressing proinsulin with the correct size in CHO-K1 cells. Immunofluorescence assay of lipopeptide transfection results shows that there is a significant difference of Corrected Total Cell Fluorescence (CTCF) between cells post-transfected with recombinant proinsulin and cells post-transfected with non-recombinant plasmid pEGFP-N1. The result is consistent with previous IFA and WB testing of cells post-transfected with recombinant proinsulin using commercial lipofection agent TurboFect™. Therefore, proinsulin has been successfully expressed in CHO-K1 cells post-transfected with recombinant proinsulin using Pal-CK2H2-Tat(NLS)-TL(NLS).

Abstrak :
[ABSTRAK
Studi cross sectional pada pasien hepatitis B di Indonesia menunjukkan korelasi mutasi kodon start pre-S2 dengan keparahan penyakit hati. Peran protein-protein HBs pada aktivasi NF-ĸB sebagai salah satu faktor dalam induksi keparahan penyakit hati. Studi ini dilakukan untuk melihat efek varian mutan HBs virus hepatitis B subgenotipe B3 sebagai strain endemik di Indonesia pada keparahan penyakit hati dilihat dari ekspresi dan aktivasi NF-ĸB. Gen HBs dari tiga pasien yang membawa tiga varian HBs berbeda diamplifikasi dan diklon dengan plasmid pcDNA3.1, ditransfeksikan dengan metode lipofektamin ke dalam sel Huh7. Nilai ekspresi mRNA dianalisis dengan real-time PCR terhadap mRNA HBs, IĸB-α, dan NF-ĸB (p50). Ekspresi IĸB-α yang diregulasi oleh NF-ĸB digunakan sebagai parameter untuk aktivasi NF-ĸB. Diperoleh plasmid ekspresi HBs dengan mutasi kodon start pre-S2, delesi pre-S2 dan wild type VHB subgenotipe B3. Plasmid rekombinan pcDNA HBs dapat mengekspresikan mRNA HBs dan menurun pada 48 hingga 72 jam. Kecuali pada mutan delesi pre-S2 yang stabil hingga 72 jam. Ekspresi protein HBs berdasar ELISA menunjukkan nilai relatif konstan pada HBs wild type, sedangkan pada HBs mutan kodon start dan delesi meningkat pada 72 jam. Aktivasi NF-ĸB relatif lebih tinggi oleh tipe wild type dibanding mutan kodon start pre-S2 dan delesi pre-S2, sehingga variasi mutasi tidak memberikan pengaruh pada aktivasi NF-ĸB, meski varian mutan delesi pre-S2 menunjukkan peningkatan aktivasi NF-ĸB setelah waktu kultur yang lebih lama dibanding HBs wild type dan mutan kodon start pre-S2. Ekspresi NF-ĸB (p50) dipengaruhi oleh variasi mutasi, ekspresi p50 lebih tinggi pada mutan kodon start pre-S2 dibanding varian HBs lainnya. Keparahan penyakit hati oleh mutasi kodon start pre-S2 dapat terkait dengan peningkatan ekspresi p50.

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ABSTRACT
Cross sectional study on hepatitis B patients in Indonesia showed association of pre-S2 start codon mutation with severity liver disease. Role of HBs proteins on the activation of NF-ĸB as one of the factor in liver disease progression. This study was to see the effects of different HBs mutant variants of Hepatitis B Virus (HBV) subgenotype B3 as the endemic strain in Indonesia on the expression and activation of NF-ĸB. HBs genes of three hepatitis B patients were amplified and cloned to pcDNA3.1, and were transfected using lipofectamine into Huh7 cell line. Expressions on mRNA level for HBs, IĸB-α and NF-ĸB (p50) were evaluated using real-time PCR. IĸB-α expression which is regulated by NF-ĸB was used as parameter to measure NF-ĸB activation. Recombinant plasmid for HBs expression with pre-S2 start codon mutation, pre-S2 deletion and wild type of HBV subgenotipe B3 were obtained. All three clones showed high level of mRNA expression which decreased after 48 to 72 hours, except for pre-S2 deletion which was relatively stabil up to72 hours. HBs protein expression detected using ELISA was constant for HBs wild type whilst increased at 72 hours for pre-S2 start codon mutation and pre-S2 deletion. NF-ĸB activation was higher for HBs wild type compared to the two mutant variants, suggesting no effect of mutation to increment of NF-ĸB activation, however pre-S2 deletion mutant showed higher NF-ĸB activation after longer period of incubation. NF-ĸB (p50) expression was higher for pre-S2 start codon mutation, suggesting liver disease progression by pre-S2 start codon mutation might associated to increased expression of p50.;Cross sectional study on hepatitis B patients in Indonesia showed association of pre-S2 start codon mutation with severity liver disease. Role of HBs proteins on the activation of NF-ĸB as one of the factor in liver disease progression. This study was to see the effects of different HBs mutant variants of Hepatitis B Virus (HBV) subgenotype B3 as the endemic strain in Indonesia on the expression and activation of NF-ĸB. HBs genes of three hepatitis B patients were amplified and cloned to pcDNA3.1, and were transfected using lipofectamine into Huh7 cell line. Expressions on mRNA level for HBs, IĸB-α and NF-ĸB (p50) were evaluated using real-time PCR. IĸB-α expression which is regulated by NF-ĸB was used as parameter to measure NF-ĸB activation. Recombinant plasmid for HBs expression with pre-S2 start codon mutation, pre-S2 deletion and wild type of HBV subgenotipe B3 were obtained. All three clones showed high level of mRNA expression which decreased after 48 to 72 hours, except for pre-S2 deletion which was relatively stabil up to72 hours. HBs protein expression detected using ELISA was constant for HBs wild type whilst increased at 72 hours for pre-S2 start codon mutation and pre-S2 deletion. NF-ĸB activation was higher for HBs wild type compared to the two mutant variants, suggesting no effect of mutation to increment of NF-ĸB activation, however pre-S2 deletion mutant showed higher NF-ĸB activation after longer period of incubation. NF-ĸB (p50) expression was higher for pre-S2 start codon mutation, suggesting liver disease progression by pre-S2 start codon mutation might associated to increased expression of p50.;Cross sectional study on hepatitis B patients in Indonesia showed association of pre-S2 start codon mutation with severity liver disease. Role of HBs proteins on the activation of NF-ĸB as one of the factor in liver disease progression. This study was to see the effects of different HBs mutant variants of Hepatitis B Virus (HBV) subgenotype B3 as the endemic strain in Indonesia on the expression and activation of NF-ĸB. HBs genes of three hepatitis B patients were amplified and cloned to pcDNA3.1, and were transfected using lipofectamine into Huh7 cell line. Expressions on mRNA level for HBs, IĸB-α and NF-ĸB (p50) were evaluated using real-time PCR. IĸB-α expression which is regulated by NF-ĸB was used as parameter to measure NF-ĸB activation. Recombinant plasmid for HBs expression with pre-S2 start codon mutation, pre-S2 deletion and wild type of HBV subgenotipe B3 were obtained. All three clones showed high level of mRNA expression which decreased after 48 to 72 hours, except for pre-S2 deletion which was relatively stabil up to72 hours. HBs protein expression detected using ELISA was constant for HBs wild type whilst increased at 72 hours for pre-S2 start codon mutation and pre-S2 deletion. NF-ĸB activation was higher for HBs wild type compared to the two mutant variants, suggesting no effect of mutation to increment of NF-ĸB activation, however pre-S2 deletion mutant showed higher NF-ĸB activation after longer period of incubation. NF-ĸB (p50) expression was higher for pre-S2 start codon mutation, suggesting liver disease progression by pre-S2 start codon mutation might associated to increased expression of p50., Cross sectional study on hepatitis B patients in Indonesia showed association of pre-S2 start codon mutation with severity liver disease. Role of HBs proteins on the activation of NF-ĸB as one of the factor in liver disease progression. This study was to see the effects of different HBs mutant variants of Hepatitis B Virus (HBV) subgenotype B3 as the endemic strain in Indonesia on the expression and activation of NF-ĸB. HBs genes of three hepatitis B patients were amplified and cloned to pcDNA3.1, and were transfected using lipofectamine into Huh7 cell line. Expressions on mRNA level for HBs, IĸB-α and NF-ĸB (p50) were evaluated using real-time PCR. IĸB-α expression which is regulated by NF-ĸB was used as parameter to measure NF-ĸB activation. Recombinant plasmid for HBs expression with pre-S2 start codon mutation, pre-S2 deletion and wild type of HBV subgenotipe B3 were obtained. All three clones showed high level of mRNA expression which decreased after 48 to 72 hours, except for pre-S2 deletion which was relatively stabil up to72 hours. HBs protein expression detected using ELISA was constant for HBs wild type whilst increased at 72 hours for pre-S2 start codon mutation and pre-S2 deletion. NF-ĸB activation was higher for HBs wild type compared to the two mutant variants, suggesting no effect of mutation to increment of NF-ĸB activation, however pre-S2 deletion mutant showed higher NF-ĸB activation after longer period of incubation. NF-ĸB (p50) expression was higher for pre-S2 start codon mutation, suggesting liver disease progression by pre-S2 start codon mutation might associated to increased expression of p50.]

Abstrak :
Pembuatan Stable Cell Line sendiri membutuhkan proses pengantaran materi genetik untuk mencapai tahap ekspresi gen rekombinan secara berkelanjutan. Metode ini menggunakan transfeksi untuk membantu mencapai tahapan tersebut. Badan Riset dan Inovasi Nasional (BRIN) Indonesia telah berhasil membuat konstruksi plasmid rekombinan pengekspresi protein Spike SARS-CoV-2. Namun, belum dilakukan penelitian lebih lanjut terkait penggunaan konstruksi plasmid rekombinan tersebut. Oleh karena itu, tujuan penelitian ini adalah untuk memvalidasi ekspresi protein rekombinan dari plasmid rekombinan pengekspresi Spike SARS-CoV-2 yang akan digunakan dalam pembuatan Stable Cell Line pada galur sel mamalia 293T. Validasi ekspresi dari empat protein SARS-CoV-2 (Spike Full, Subunit S1, Subunit S2, dan Receptor Binding Domain) dilakukan melalui metode Immunofluorescence Assay (IFA) dan Western Blot (WB). Hasil menunjukkan bahwa dari empat ragam protein (Spike Full, Subunit S1, Subunit S2, dan Receptor Binding Domain) terbukti fungsional secara ekspresi dan sesuai dengan ukuran protein yang sesuai. Uji IFA menunjukkan bahwa terdapat dua nilai rata-rata Corrected Total Cell Fluorescence (CTCF) yang unggul yaitu pada protein Spike Full dan Subunit S2 (118.813 dan 264.159 CTCF) sel pasca transfeksi yang menandakan bahwa terdapat perbedaan kemampuan ekspresi dari masing-masing protein. Uji western blot telah membuktikan dua protein (Spike Full dan Subunit S2) memiliki ukuran molekul yang sesuai (142,5 dan 66,0 kDa). Sehingga, dapat disimpulkan bahwa plasmid rekombinan yang dikonstruksi oleh BRIN terbukti fungsional dan dapat dilanjutkan ke dalam penggunaannya untuk pembuatan Stable Cell Line. ......Making a Stable Cell Line requires a process of delivering genetic material to reach the continuous recombinant gene expression stage. This method uses transfection to help achieve this stage. The National Research and Innovation Agency of Indonesia (BRIN) has successfully constructed a recombinant plasmid expressing the SARS-CoV-2 Spike protein. However, no further research has been carried out regarding using these recombinant plasmid constructs. Therefore, this study aimed to validate the expression of recombinant proteins from the SARS-CoV-2 Spike-expressing recombinant plasmid to manufacture Stable Cell Line in mammalian cell line 293T. Validation of the expression of four SARS-CoV-2 proteins (Spike Full, Subunit S1, Subunit S2, and Receptor Binding Domain) was carried out using Immunofluorescence Assay (IFA) and Western Blot (WB) methods. The results showed that the four protein variants (Spike Full, S1 Subunit, S2 Subunit, and Receptor Binding Domain) were functional in expression and according to the appropriate protein size. The IFA test showed two superior Corrected Total Cell Fluorescence (CTCF) values: the Spike Full protein and the S2 Subunit (118.813 and 264159 CTCF) of post-transfection cells, which indicated that there were differences in the expression ability of each protein. The Western Blot test has proven that two proteins (Spike Full and Subunit S2) have the appropriate molecular size (142.5 and 66.0 kDa). Thus, it can be concluded that the recombinant plasmid constructed by BRIN is proven to be functional and can be used to manufacture Stable Cell Lines.